Altered Gene Expression in Three Plant Species inResponse to Treatment with Nep1, a Fungal Protein ThatCauses Necrosis
Sarah E. Keates, Todd A. Kostman, James D. Anderson, and Bryan A. Bailey*
Alternate Crops and Systems Laboratory, U.S. Department of Agriculture/Agricultural Research Service,Beltsville Area Research Center-West, Beltsville, Maryland 20705 (S.E.K., B.A.B.); Department of Biology andMicrobiology, University of Wisconsin, Oshkosh, Wisconsin 54901 (T.A.K.); and Plant Sciences Institute,Beltsville Area Research Center-West, Beltsville, Maryland 20705 (J.D.A.)
Nep1 is an extracellular fungal protein that causes necrosis when applied to many dicotyledonous plants, including invasiveweed species. Using transmission electron microscopy, it was determined that application of Nep1 (1.0 �g mL�1, 0.1% [v/v]Silwet-L77) to Arabidopsis and two invasive weed species, spotted knapweed (Centaurea maculosa) and dandelion (Tarax-acum officinale), caused a reduction in the thickness of the cuticle and a breakdown of chloroplasts 1 to 4 h after treatment.Membrane breakdown was most severe in cells closest to the surface of application. Differential display was used to isolatecDNA clones from the three species showing differential expression in response to Nep1 treatment. Differential geneexpression was observed for a putative serpin (CmSER-1) and a calmodulin-like (CmCAL-1) protein from spotted knap-weed, and a putative protein phosphatase 2C (ToPP2C-1) and cytochrome P-450 (ToCYP-1) protein from dandelion. Inaddition, differential expression was observed for genes coding for a putative protein kinase (AtPK-1), a homolog (AtWI-12)of wound-induced WI12, a homolog (AtLEA-1) of late embryogenesis abundant LEA-5, a WRKY-18 DNA-binding protein(AtWRKY-18), and a phospholipase D (AtPLD-1) from Arabidopsis. Genes showing elevated mRNA levels in Nep1-treated(5 �g mL�1, 0.1% [v/v] Silwet-L77) leaves 15 min after Nep1 treatment included CmSER-1 and CmCAL-1 for spottedknapweed, ToCYP-1 and CmCAL-1 for dandelion, and AtPK-1, AtWRKY-18, AtWI-12, and AtLEA-1 for Arabidopsis. Levelsof mRNA for AtPLD-1 (Arabidopsis) and ToPP2C-1 (dandelion) decreased rapidly in Silwet-L77-treated plants between 15min and 4 h of treatment, but were maintained or decreased more slowly over time in Nep1-treated (5 �g mL�1, 0.1% [v/v]Silwet-L77) leaves. In general, increases in mRNA band intensities were in the range of two to five times, with only ToCYP-1in dandelion exceeding an increase of 10 times. The identified genes have been shown to be involved or are related to genefamilies that are involved in plant stress responses, including wounding, drought, senescence, and disease resistance.
The necrosis-inducing protein Nep1 is an extracel-lular protein produced by Fusarium oxysporum in liq-uid cultures (Bailey, 1995; Bailey et al., 1997). Thegene for Nep1 has been cloned (GenBank accessionno. AF036580) and partially characterized (Nelson etal., 1998; Bailey et al., 2002). Antigenically relatedproteins of similar size and activity are produced byFusarium accuminatum and Fusarium avenaceum(Bailey et al., 1997). Subsequent to the original isola-tion and characterization of NEP1, related gene andgene products have been identified (GenBank) asbeing produced by species of Pythium, Streptomyces,Phytophthora, and other genera, which demonstratesthat Nep1-related proteins are widely produced bytaxonomically divergent microorganisms. Nep1 pro-duction was disrupted in F. oxysporum f. sp. eryth-roxyli using gene replacement, thus providing evi-dence that Nep1 production is not critical todevelopment of Fusarium wilt of Erythroxylum coca(Bailey et al., 2002).
Nep1 kills cells of many different dicotyledonousplants but does not damage monocots (Bailey, 1995;Jennings et al., 2000). Nep1 defoliates some dicotyle-donous weed species in a manner similar to a contactherbicide when applied as a foliar spray (Bailey et al.,2000b; Jennings et al., 2000). Nep1 can be combinedwith the herbicides 2,4-dichlorophenoxyacetic acid orglyphosphate (Bailey et al., 2000b). The application ofthe Nep1-herbicide combinations to spotted knap-weed (Centaurea maculosa) resulted in rapid foliarnecrosis caused by Nep1 followed by eventual deathof the weed caused by the herbicides (Bailey et al.,2000b). It has also been demonstrated that Nep1 canbe applied in combination with the plant pathogenPleospora papaveracea, which results in enhanced dis-ease development and death of opium poppy (Papav-er somniferum; Bailey et al., 2000a).
In addition to necrosis, tobacco (Nicotiana tabacum)cellular responses to Nep1 treatment include ethyl-ene production, active oxygen production, alteredcell respiration, K� and H� channel fluxes, and al-tered gene expression (Jennings et al., 2001). Nep1induces many of the same processes characteristic ofelicitors of plant defense responses in plants (Jen-nings et al., 2000). The protein has not been demon-
strated to induce resistance to plant disease andtherefore cannot definitively be distinguished frommembrane-altering toxins (Jennings et al., 2000). Atpresent, it is unclear what processes allow Nep1 toact as a contact herbicide that promotes plant diseaseinstead of plant defense. The objectives of this re-search are to begin characterizing the responses ofthe Arabidopsis and the invasive weeds spottedknapweed and dandelion (Taraxacum officinale) toNep1 at the cellular level using transmission electronmicroscopy and at the molecular level using differ-ential display. The knowledge gained should helpour understanding of how Nep1 acts on plant cellsand should provide clues as to how Nep1 can beexploited as a biologically produced herbicide.
RESULTS
The Necrotic Response to Nep1
Spotted knapweed and dandelion were much moresensitive to Nep1 than Arabidopsis was in thesestudies. Spotted knapweed and dandelion had 25%and 75% leaf necrosis 24 h after treatment with 1 and5 �g mL�1 Nep1 (plus 0.1% [v/v] Silwet-L77), re-spectively. Necrotic spots on spotted knapweed anddandelion were uniformly spread and coalesced,sometimes covering whole leaves by 48 h after treat-ment. Arabidopsis had only 3% necrosis after treat-ment with 5 �g mL�1 Nep1 (plus 0.1% [v/v] Silwet-L77), and necrosis caused by the 1 �g mL�1 Nep1(plus 0.1% [v/v] Silwet-L77) was not measurablemacroscopically on Arabidopsis. Necrosis on Arabi-dopsis was observed as a tip burn that did not de-velop further after 24 h. The Silwet-L77 treatment(0.1%, v/v) did not cause measurable necrosis on anyof the three plant species.
Transmission Electron Microscopy
Application of Nep1 (1 �g mL�1, 0.1% [v/v]Silwet-L77) caused a noticeable decrease in the thick-ness of the leaf cuticle between 1 and 4 h after treat-ment (Figs. 1 and 2). Changes in dandelion (data notshown) and Arabidopsis (Fig. 2) were apparent at the1- and 4-h time points, and changes in spotted knap-weed were observed at the 4-h time point (Fig. 1).The cuticle appears in micrographs as a darker layeron the upper (abaxial) surface of epidermal cell walls.The dark staining is due to the presence of waxes andesters in the cuticle that react with the osmium tet-roxide used as a postfixative on these samples. Thedecrease in cuticle thickness took on two forms. Inthe treated spotted knapweed leaves, the cuticle ap-peared to be degrading and “sloughing off” (as indi-cated by arrows), compared with the cuticle in theSilwet-L77-treated (0.1%, v/v) leaf, which remained asolid dark gray layer (Fig. 1B, arrows). In dandelion(data not shown) and Arabidopsis (Fig. 2B), the cu-ticle (as indicated by arrows) of Nep1-treated (1 �g
mL�1, 0.1% [v/v] Silwet-L77) leaf tissues appeared tobe compressed to a thinner, more darkly stainedlayer. This is in comparison with the cuticles inSilwet-L77-treated (0.1%, v/v) tissues, where the cu-ticle was thicker and more lightly stained (Fig. 2A).
Chloroplasts withstood the fixation process betterthan most other organelles, allowing their detaileddescription. Data concerning the responses of otherorganelles and membranes were inconclusive. Nep1(1 �g mL�1, 0.1% [v/v] Silwet-L77) had a markedeffect on the appearance of chloroplasts and chloro-plast membranes in all three species studied (Figs. 1and 2). In general, application of Nep1 caused break-down of the thylakoid and granal membrane struc-tures that was noticeable in dandelion (data notshown) and Arabidopsis (Fig. 2D) at the 1- and 4-htime points; changes in spotted knapweed (Fig. 1D)were observed at the 4-h time point. Also noticeable
Figure 1. Nep1-induced changes in cuticle and chloroplasts of spot-ted knapweed as detected by transmission electron microscopy. Athrough D, Transmission electron micrographs of adaxial epidermaland mesophyll cells before and after application of Nep1. A, Cuticlein control leaf, arrow points to cuticle layer. B, Cuticle layer (arrow)in leaf 4 h after application of Nep1. Note degradation of cuticle(lighter gray areas). C, Chloroplast in untreated (control) leaf. Arrowspoint to normal granal stacks and thylakoid membranes. D, Chloro-plast in leaf 4 h after treatment with Nep1. Note lack of organizedgranal stacks and thylakoid membranes (arrows). Starch grains seemto be breaking down; also of interest are the many lipophilic bodiespresent. Bars � 0.4 �M in A and B, and 1 �M in C and D. sg, starchgrain.
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was the disappearance of starch granules from chlo-roplasts of treated leaves (Fig. 2D). This is of notebecause all of the leaves were harvested at the sametime of day and under the same light conditions. Thechloroplasts themselves also appeared to swell andbecome misshapen after treatment with Nep1 (1 �gmL�1, 0.1% [v/v] Silwet-L77), and the outer chloro-plast membrane appeared to deteriorate, which wasespecially evident in Arabidopsis (Fig. 2D). Last, chlo-roplasts from Nep1-treated (plus 0.1% [v/v] Silwet-L77) leaves often appeared to have more lipophilicbodies compared with chloroplasts from Silwet-L77-treated (0.1%, v/v) leaves (Figs. 1D and 2D). Note thatall of the observations above pertain to chloroplastsnearer the point of Nep1 application. Chloroplasts onthe opposite side of the leaf from the application siteshowed no change in appearance (data not shown).
Isolation and Identification of Putatively DifferentiallyExpressed cDNA Clones
Taking into account the three plant species beingstudied, a total of 29 cDNA fragments were cloned
and sequenced based on their consistent differentialexpression on two replicate differential display gels.Of the 29 clones, 15 showed no significant homologyto known sequences using blastx and tblastx. Nine ofthe 15 unidentified clones were isolated from spottedknapweed and five were isolated from dandelion.Two clones, one from spotted knapweed and onefrom dandelion, showed homology to unknown Ara-bidopsis proteins. The remaining 11 clones (Table I),which showed significant homology to known genesor gene families, were selected for further study.
Eleven cDNA clones (Table I) were used as probeson northern blots of total RNA from Silwet-L77- (1%,v/v) and Nep1-treated (1 �g mL�1, 0.1% [v/v]Silwet-L77) leaves for the three plant species beingstudied without consideration of the origin of theclone. Only CmTIF-1 and CmCAL-1 hybridized tototal RNA from plant species other than the speciesfrom which the clone was obtained (data not shown).CmTIF-1 hybridized to total RNA from all three spe-cies but did not show differential expression, andCmCAL-1 hybridized with total RNA from spottedknapweed and dandelion showing differential ex-pression in both species. Only the cDNA clone/plantspecies combinations that showed significant hybrid-ization to northern blots in the studies of the 1 �gmL�1 Nep1 (plus 0.1% [v/v] Silwet-L77) treatmentswere used when probing northern blots of total RNAfrom leaves treated with 5 �g mL�1 Nep1 (plus 0.1%[v/v] Silwet-L77). Unless otherwise indicated, theresults for studies using the 1 �g mL�1 Nep1 rate(plus 0.1% [v/v] Silwet-L77) were similar to thosepresented for the 5 �g mL�1 Nep1 rate (plus 0.1%[v/v] Silwet-L77), and the data are not shown. CmB-Glu-1 showed low level induction in Silwet-L77-treated (0.1%, v/v) spotted knapweed 4 h after treat-ment (data not shown) and was not studied further.
Gene Expression
Spotted Knapweed
The quantity of CmCAL-1 mRNA present onnorthern blots of total RNA (20 �g lane�1) extractedfrom Silwet-L77-treated (1%, v/v) spotted knapweedleaves decreased to almost undetectable levels be-tween 15 min and 1 h after treatment (Fig. 3). TotalRNA (20 �g lane�1) isolated from spotted knapweedleaves 15 min after treatment with Nep1 (5 �g mL�1,1% [v/v] Silwet-L77) had two times more CmCAL-1mRNA than Silwet-L77 samples at the same timepoint. CmCAL-1 mRNA quantities were maintainedfor 4 h in Nep1-treated spotted knapweed leaves.Four hours after treatment, 5.2 times more CmCAL-1mRNA was detected on northern blots of total RNAfrom Nep1-treated spotted knapweed leaves com-pared with northern blots of total RNA from Silwet-L77-treated spotted knapweed leaves.
Total RNA (20 �g lane�1) extracted from spottedknapweed leaves after treatment with Nep1 (5 �g
Figure 2. Nep1-induced changes in cuticle and chloroplasts of Ara-bidopsis as detected by transmission electron microscopy. A throughD, Transmission electron micrographs of adaxial epidermal and me-sophyll cells. A, Cuticle of untreated leaf (as indicated by arrows). B,Cuticle of leaf 1 h after treatment with Nep1. Note decrease inthickness and dark appearance. C, Chloroplasts in mesophyll cell ofuntreated leaf. Note normal membrane structure and presence ofstarch grains. D, Chloroplast in mesophyll cell of treated leaf. Notelack of organized membrane structure, altered starch grains, and mis-shapen nature of chloroplast. Bars � 1 �M. cw, Cell wall; sg, starchgrain.
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mL�1 Nep1, 1% [v/v] Silwet-L77) had approximatelytwo times more CmSER-1 mRNA than samples ex-tracted from Silwet-L77-treated (1%, v/v) leaves atall time points sampled (Fig. 4). The quantity ofCmSER-1 mRNA in Silwet-L77-treated spotted knap-weed leaves remained steady at 15 min, 1 h, and 4 h.
Dandelion
The quantity of mRNA CmCAL-1 on northern blotsof total RNA (20 �g lane�1) extracted from Silwet-L77-treated (1%, v/v) dandelion leaves decreased toalmost undetectable levels between 1 and 4 h aftertreatment (Fig. 5). Total RNA (20 �g lane�1) isolatedfrom dandelion leaves 15 min after treatment withNep1 (5 �g mL�1, 1% [v/v] Silwet-L77) had 2.4 timesmore CmCAL-1 mRNA than Silwet-L77 samples atthe same time point. CmCAL-1 mRNA quantitieswere maintained for 4 h in Nep1-treated dandelionleaves. As a result, 5.8 times more CmCAL-1 mRNAwas detected on northern blots of total RNA fromNep1-treated dandelion leaves compared with north-ern blots of total RNA from Silwet-L77-treated dan-delion leaves 4 h after treatment.
ToCYP-1 mRNA accumulated in response to Nep1treatment. The Nep1 rate strongly influenced thetime course of ToCYP-1 mRNA accumulation (Fig. 6).Fifteen minutes after treatment, total RNA from the 5
�g mL�1 Nep1-treated (plus 0.1% [v/v] Silwet-L77)dandelion leaves had 11 times more ToCYP-1 mRNAthan Silwet-L77-treated (1%, v/v) dandelion leaves.The high level of ToCYP-1 mRNA was maintainedfor 4 h. Treatment of dandelion with 1 �g mL�1 Nep1(plus 0.1% [v/v] Silwet-L77) resulted in ToCYP-1mRNA accumulation at 1 and 4 h but not at 15 min.
The quantity of ToPP2C-1 mRNA detected onnorthern blots of total RNA isolated from Nep1-treated (5 �g mL�1, 1% [v/v] Silwet-L77) dandelionleaves remained steady between 15 min and 4 h aftertreatment (Fig. 7). The quantity of ToPP2C-1 mRNAdetected decreased on northern blots of total RNAisolated from Silwet-L77-treated (1%, v/v) dandelionleaves between 15 min and 4 h. As a result, six timesmore ToPP2C-1 mRNA was detected on northernblots of total RNA from Nep1-treated dandelionleaves compared with northern blots of total RNAfrom Silwet-L77-treated dandelion leaves 4 h aftertreatment.
Arabidopsis
Fifteen minutes after treatment, 4.8 times more pro-tein AtPK-1 mRNA was detected on northern blots oftotal RNA (20 �g lane�1) extracted from Nep1-treated (5 �g mL�1, 1% [v/v] Silwet-L77) Arabidop-sis leaves compared with northern blots of total RNA
Table I. Putative Nep1c responsive cDNA clones isolated from spotted knapweed, dandelion, or Arabidopsis using differential display andtheir tentative identification based on sequence comparisons (BLASTX)
from Silwet-L77-treated (1%, v/v) Arabidopsisleaves (Fig. 8). AtPK-1 mRNA levels decreased be-tween 15 min and 4 h after Nep1 treatment. AtPK-1mRNA levels increased between 15 min and 1 h aftertreatment with Silwet-L77 to the point that there waslittle difference between the Nep1 and Silwet-L77treatments 1 and 4 h after treatment.
Fifteen minutes after treatment, 4.3 times moreAtWRKY-18 mRNA was detected on northern blotsof total RNA (20 �g lane�1) extracted from Nep1-treated (5 �g mL�1, 1% [v/v] Silwet-L77) Arabidop-sis leaves compared with northern blots of total RNAfrom Silwet-L77-treated (0.1%, v/v) Arabidopsisleaves (Fig. 9). AtWRKY-18 mRNA levels decreasedbetween 15 min and 4 h after Nep1 treatment.AtWRKY-18 mRNA levels were most variable 1 hafter treatment for Nep1- and Silwet-L77-treated Ara-bidopsis leaves.
Fifteen minutes after treatment, 1.8 times moreAtPLD-1 mRNA was detected on northern blots oftotal RNA (20 �g lane�1) extracted from Silwet-L77-treated (1%, v/v) Arabidopsis leaves compared withnorthern blots of total RNA from Nep1-treated (5 �g
mL�1, 1% [v/v] Silwet-L77) Arabidopsis leaves (Fig.10). AtPLD-1 transcript decreased in Silwet-L77-treated Arabidopsis leaves to almost undetectablelevels 1 h after treatment. AtPLD-1 mRNA decreasedmore slowly in Nep1-treated Arabidopsis leaves be-tween 15 min and 4 h. As a result, AtPLD-1 mRNAlevels for samples from Nep1-treated Arabidopsisleaves were at least 1.7 times that detected for sam-ples from Silwet-L77-treated leaves 1 and 4 h aftertreatment.
When AtWI-12 and AtLEA-1 were used as probeson northern blots, they produced similar expressionprofiles (Figs. 11 and 12). Fifteen minutes and 1 hafter treatment, AtWI-12 and AtLEA-1 mRNA levelsfor samples from Nep1-treated (5 �g mL�1, 1% [v/v]Silwet-L77) Arabidopsis leaves were at least threetimes that observed for samples from Silwet-L77-treated (0.1%, v/v) Arabidopsis leaves. The mRNAlevels for AtWI-12 and AtLEA-1 decreased between 1and 4 h after Nep1 treatment.
Figure 3. Expression of CmCAL-1 (calmodulin-like) in spotted knap-weed after treatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N).Spotted knapweed was treated with Silwet-L77 (0.1%, v/v) or 5 �gmL�1 Nep1 (plus 0.1% [v/v] Silwet-L77). Leaves were harvested 15,60, or 240 min after treatment. A, Representative northern blot (20�g total RNA per lane). B, Corrected band volumes for each treat-ment by time combination (TXT) were converted to ratios relative tothe 15-min Silwet-L77 sample (S15) on each blot. The ratios (TXT/S15) were averaged for each treatment at each time point using datafor three replicate blots.
Figure 4. Expression of CmSER-1 (serpin-like) in spotted knapweedafter treatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N).Spotted knapweed was treated with Silwet-L77 (0.1%, v/v) or 5 �gmL�1 Nep1 (plus 0.1% [v/v] Silwet-L77). Leaves were harvested 15,60, or 240 min after treatment. A, Representative northern blot (20�g total RNA per lane). B, Corrected band volumes for each treat-ment by time combination (TXT) were converted to ratios relative tothe 15-min Silwet-L77 sample (S15) on each blot. The ratios (TXT/S15) were averaged for each treatment at each time point using datafor three replicate blots.
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DISCUSSION
Structural Changes Caused by Nep1
Plant cells respond to Nep1 by rapid structuralchanges, including the thinning of the cuticle anddisruption of chloroplasts. Similar changes could beseen by transmission electron microscopy within 1 hof Nep1 treatment in all three species studied, longbefore macroscopic necrosis was observed. Arabi-dopsis was less sensitive to Nep1 at the macroscopiclevel, but the types of structural changes caused byNep1 to Arabidopsis closely resembled changes ob-served in Nep1-treated dandelion.
The chloroplast changes we observed included,most strikingly, a complete breakdown of the inter-nal chloroplast membranes, breakdown of the outerchloroplast membrane, loss of starch grains, and a“swelling” of the chloroplasts. In studies of the hy-persensitive response in plant cells, similar changesto chloroplasts and other cell membrane structureshave been observed (Goodman, 1972; Meyer andHeath, 1988a). What is unique about our observa-tions is the fact that they are also at the subcellular
Figure 5. Expression of CmCAL-1 (calmodulin-like) in dandelionafter treatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N).Dandelion was treated with Silwet-L77 (0.1%, v/v) or 5 �g mL�1
Nep1 (plus 0.1% [v/v] Silwet-L77). Leaves were harvested 15, 60, or240 min after treatment. A, Representative northern blot (20 �g totalRNA per lane). B, Corrected band volumes for each treatment by timecombination (TXT) were converted to ratios relative to the 15-minSilwet-L77 sample (S15) on each blot. The ratios (TXT/S15) wereaveraged for each treatment at each time point using data for threereplicate blots.
Figure 6. Expression of ToCYP-1 (cytochrome P450) in dandelionafter treatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N).Dandelion was treated with Silwet-L77 (0.1%, v/v) or (A and B) 5 �gmL�1 Nep1 (plus 0.1% [v/v] Silwet-L77) or (C and D) 1 �g mL�1
Nep1 (plus 0.1% [v/v] Silwet-L77). Leaves were harvested 15, 60, or240 min after treatment. A and C, Representative northern blot (20 �gtotal RNA per lane). B and D, Corrected band volumes for eachtreatment by time combination (TXT) were converted to ratios rela-tive to the 15-min Silwet-L77 sample (S15) on each blot. The ratios(TXT/S15) were averaged for each treatment at each time point usingdata for three replicate blots.
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level, and followed application of a purified fungalelicitor protein to intact leaves, whereas in otherstudies of this kind, leaf necrosis has only been de-scribed using naked eye and light microscopy (Ricciet al., 1989; Heath, 1997; Roussel et al., 1999; Ivanovaet al., 2001). It is also interesting to note that thebreakdown of chloroplast membranes that we ob-served was concurrent with the appearance of manyplastoglobuli, a phenomenon associated with mem-brane breakdown in chloroplasts of senescing leaves(Goodman et al., 1986).
The rapid (1–4 h) changes in cuticle thickness inresponse to Nep1 treatment were unexpected andraise the possibility that Nep1 directly acts on thecuticle. The change in cuticle thickness in response toNep1 was consistent across the three species and atmultiple time points. Many compounds within thecuticle, such as the epicuticular waxes (Eglinton andHamilton, 1967; Jetter and Schaffer, 2001), are li-pophylic in nature. Nep1 also rapidly alters cellmembranes (Jennings et al., 2001) and lipid bilayers,supporting the possibility that Nep1 interacts di-rectly with lipophylic compounds as has been dem-onstrated for toxins and elicitors (Lee et al., 2001).
Alternatively, it is possible that Nep1 indirectlyaffects cuticle thickness as a result of other rapidlyinduced changes in plant cells. Tobacco cell suspen-sions, which lack a cuticle, respond to Nep1 within 15min by electrolyte leakage and production of activeoxygen, and subsequently die (Jennings et al., 2001),indicating that action on the cuticle is not a prereq-uisite for Nep1-induced cell death. It has been dem-onstrated that treating opium poppy plants withNep1 makes the plants more susceptible to infectionby P. papaveracea (Bailey et al., 2000a). Regardless ofthe mode of action, a thinner cuticle layer might beeasier for fungal pathogens to penetrate, causingNep1-treated plants to be more susceptible todisease.
Genes Potentially Involved Early inSignal Transduction
AtPK-1 in Arabidopsis and ToPP2C-1 in dandelionputatively code for a protein kinase and a proteinphosphatase 2C, respectively (Table I). Reversibleprotein phosphorylation is mediated by protein ki-
Figure 7. Expression of ToPP2C-1 (protein phosphatase) in dande-lion after treatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N).Dandelion was treated with Silwet-L77 (0.1%, v/v) or 5 �g mL�1
Nep1 (plus 0.1% [v/v] Silwet-L77). Leaves were harvested 15, 60, or240 min after treatment. A, Representative northern blot (20 �g totalRNA per lane). B, Corrected band volumes for each treatment by timecombination (TXT) were converted to ratios relative to the 15-minSilwet-L77 sample (S15) on each blot. The ratios (TXT/S15) wereaveraged for each treatment at each time point using data for threereplicate blots.
Figure 8. Expression of AtPK-1 (protein kinase) in Arabidopsis aftertreatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N). Arabidop-sis was treated with Silwet-L77 (0.1%, v/v) or 5 �g mL�1 Nep1 (plus0.1% [v/v] Silwet-L77). Leaves were harvested 15, 60, or 240 minafter treatment. A, Representative northern blot (20 �g total RNA perlane). B, Corrected band volumes for each treatment by time com-bination (TXT) were converted to ratios relative to the 15-min Silwet-L77 sample (S15) on each blot. The ratios (TXT/S15) were averagedfor each treatment at each time point using data for three replicateblots.
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nases and protein phosphatases and serves as a pri-mary regulator of many signal transduction path-ways (Ganguly and Singh, 1999). AtPK-1 is closelyrelated (91% identical) to the Ser/Thr-specific proteinkinase NAK in Arabidopsis (Moran and Walker,1993). NAK is similar to the oncogenes met (Park etal., 1987) and abl (Daniel et al., 2001), and based onthe homology they share with other protein kinases,may play a role in the regulation of plant growth anddevelopment (Moran and Walker, 1993). ToPP2C-1 isclosely related to a protein phosphatase 2C of Arabi-dopsis, PP2C5 (Table I), and a protein phosphatase2C of alfalfa, PP2C (Table I). PP2C5 is regulated byabscisic acid (Wang et al., 1999). PP2C is induced bycold, drought, touch, and wounding, and it functionsas a negative regulator of the stress-activatedmitogen-activated protein kinase pathway in plants(Meskiene et al., 1998). Hyperphosphorylation is be-lieved to be involved in events leading to enhancedethylene production and senescence in flower of Pha-laenopsis spp. (Wang et al., 2001) and elicitor-inducedreactive oxygen species generation, H� influx, and
cytoplasmic acidification in suspension-cultured rice(Oryza sativa) cells (He et al., 1998).
AtWRKY-18 is the gene for a salicylic acid/pathogen-induced WRKY DNA-binding protein inArabidopsis (Table I). The WRKY DNA-binding pro-teins make up a superfamily of transcription factorsthat are involved in the regulation of gene expres-sion, including genes involved in plant defense andsenescence (Du and Chen, 2000; Eulgem et al., 2000;Hara et al., 2000). AtWRKY-18 was highly induced inArabidopsis 1 to 8 h after salicylic acid treatment (Yuet al., 2001), whereas induction of AtWRKY-18 was ata maximum 15 min after Nep1 treatment (Fig. 9).NPR1, a positive regulator of inducible plant diseaseresistance in Arabidopsis, has W-box sequences in itspromoter region that are recognized by theAtWRKY-18 protein (Yu et al., 2001).
CmCAL-1 was induced by Nep1 treatment in spot-ted knapweed and dandelion. CmCAL-1 is closelyrelated to genes for calmodulin-like proteins in Ara-bidopsis and many other plant species, but did nothybridize to northern blots of total RNA from Nep1-treated Arabidopsis under the conditions used. Cal-modulin and calmodulin-related genes in Arabidop-
Figure 9. Expression of AtWRKY-18 (WRKY-18) in Arabidopsis aftertreatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N). Arabidop-sis was treated with Silwet-L77 (0.1%, v/v) or 5 �g mL�1 Nep1 (plus0.1% [v/v] Silwet-L77). Leaves were harvested 15, 60, or 240 minafter treatment. A, Representative northern blot (20 �g total RNA perlane). B, Corrected band volumes for each treatment by time com-bination (TXT) were converted to ratios relative to the 15-min Silwet-L77 sample (S15) on each blot. The ratios (TXT/S15) were averagedfor each treatment at each time point using data for three replicateblots.
Figure 10. Expression of AtPLD-1 in Arabidopsis after treatment withSilwet-L77 (S) or Nep1 plus Silwet-L77 (N). Arabidopsis was treatedwith Silwet-L77 (0.1%, v/v) or 5 �g mL�1 Nep1 (plus 0.1% [v/v]Silwet-L77). Leaves were harvested 15, 60, or 240 min after treat-ment. A, Representative northern blot (20 �g total RNA per lane). B,Corrected band volumes for each treatment by time combination(TXT) were converted to ratios relative to the 15-min Silwet-L77sample (S15) on each blot. The ratios (TXT/S15) were averaged foreach treatment at each time point using data for three replicate blots.
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sis and other plant species are regulated by physicalinducers, including rain, wind, touch, and wound-ing, resulting in rapid transcript accumulation andaltered development (Braam and Davis, 1990;Takezawa et al., 1995). Leshem (1984) proposed theinvolvement of calcium/calmodulin in senescence asmediated by the action of ethylene, free radicals,lipooxygenase, and phospholipase A2. Calcium/cal-modulin complexes can activate protein kinases thatare important in signal transduction pathways (Vo-geli et al., 1992) and that are involved in induction ofphytoalexin biosynthesis by cell wall elicitors (Vogeliet al., 1992).
Signaling cascades can be triggered by the activa-tion of phospholipid-cleaving enzymes such as phos-pholipases C, D (PLD), and A(2) (Qin et al., 1997).AtPLD-1 (Arabidopsis) putatively codes for a phos-pholipase D (PLD) � (Table I). AtPLD-1 mRNA de-crease rapidly with time after Silwet-L77 treatmentscompared with a slower mRNA decrease after Nep1(plus Silwet-L77) treatment, which suggests regula-tion by multiple factors. AtPLD-1 is closely related toAtPLD� (97% identity), a PLD�. AtPLD� enzyme
activity is dependent on phosphatidylinositol 4,5-bisphosphate and nanomolar concentrations of cal-cium (Pappan et al., 1997). PLD�1 mRNA was foundto rapidly and specifically accumulate in tomato (Ly-copersicon esculentum) cell suspensions and tomatoleaves treated with a fungal elicitor xylanase (Laxaltet al., 2001). In the resurrection plant, Craterostigmaplantagineum, CpPLD-2 coding for a PLD was in-duced by dehydration and abscisic acid (Frank et al.,2000).
Other Genes Potentially Involved in PlantResponses to Stress
Nep1 (5 �g mL�1, 0.1% [v/v] Silwet-L77) inducesaccumulation of ToCYP-1 transcript in dandelionwithin 15 min of treatment and is the most highlyup-regulated of the genes studied. ToCYP-1 puta-tively codes for a cytochrome P450 protein (Table I).Cytochrome P450 enzymes form a large superfamilyof genes (Nelson et al., 1993) that can be induced inplants by many different stimuli, including chemi-cals, elicitors, wounding, pathogen attack, and other
Figure 11. Expression of AtWI-12 (wound-induced protein) in Ara-bidopsis after treatment with Silwet-L77 (S) or Nep1 plus Silwet-L77(N). Arabidopsis was treated with Silwet-L77 (0.1%, v/v) or 5 �gmL�1 Nep1 (plus 0.1% [v/v] Silwet-L77). Leaves were harvested 15,60, or 240 min after treatment. A, Representative northern blot (20�g total RNA per lane). B, Corrected band volumes for each treat-ment by time combination (TXT) were converted to ratios relative tothe 15-min Silwet-L77 sample (S15) on each blot. The ratios (TXT/S15) were averaged for each treatment at each time point using datafor three replicate blots.
Figure 12. Expression of AtLEA-1 (late embryogenesis abundant) inArabidopsis after treatment with Silwet-L77 (S) or Nep1 plus Silwet-L77 (N). Arabidopsis was treated with Silwet-L77 (0.1%, v/v) or 5 �gmL�1 Nep1 (plus 0.1% [v/v] Silwet-L77). Leaves were harvested 15,60, or 240 min after treatment. A, Representative northern blot (20�g total RNA per lane). B, Corrected band volumes for each treat-ment by time combination (TXT) were converted to ratios relative tothe 15-min Silwet-L77 sample (S15) on each blot. The ratios (TXT/S15) were averaged for each treatment at each time point using datafor three replicate blots.
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stress factors (Schopfer and Ebel, 1998; Oh et al.,1999; Takemoto et al., 1999). ToCYP-1 is most closelyrelated to PepCYP (49% identical) from peppers (Cap-sicum annuum), based on partial sequence compari-sons (Table I). PepCYP is a cytochrome P450 proteinthat is highly expressed in an incompatible interac-tion of pepper to Colletotrichum gloeosporioides and isup-regulated by wounding or jasmonic acid treat-ment (Oh et al., 1999). Cytochrome P450 enzymes cancatalyze oxidative reactions that function to detoxifycompounds. In addition, cytochrome P450 enzymescan function in the biosynthesis of compounds in-cluding phytoalexins.
AtWI-12 is related to an elicitor inducible protein intobacco (59% identity) and the wound-induced pro-tein WI12 in common ice plant (Mesembryanthemumcrystallinum; 51% identity). Wounding, methyl jas-monate, and pathogen infection induced local WI12expression in common ice plant (Yen et al., 2001).AtLEA-1 shows similarity (58%) to the gene codingfor the late embryogenesis abundant protein Lea5 intobacco (Weaver et al., 1998). Members of the LEAgenes family have been associated with plant re-sponses to many different stresses includingdrought, salt, cold, heat, wounding, and several planthormones in addition to natural senescence (Weaveret al., 1998), but the function of LEA family membersin plants remains obscure.
CmSER-1 is most closely related to serpin genesfound in barley (Table I), wheat (Triticum aestivum),Avena fatua, and Cucurbita maxima in addition to pu-tative serpins found in Arabidopsis (Table I). Serpins,Ser proteinase inhibitors, are well described in higheranimals where they function in divergent biologicalprocesses (Silverman et al., 2001). In plants, serpinsare best described in barley and wheat where theyare found as abundant endosperm proteins. Al-though protease inhibitors in many cases have beenshown to function in the defense response of plantsto chewing insects (Lawrence and Koundal, 2002),the function of serpins in plant biology is unclear(Silverman et al., 2001). Evidence for induction ofserpin genes in response to stress is lacking in plants.CmPS-1, a serpin found in C. maxima phloem and iscorrelated with increased resistance to the aphid My-zus pericae in vivo, is developmentally regulated, andhas activity as an elastase inhibitor (Yoo et al., 2000).
Summary
It remains unclear if the identified responses toNep1 treatment should be characterized as a generalstress response to a toxin or some form of inducedresistance (Jennings et al., 2001). Many plant genesare induced by pathogens and/or microbial elicitors,and their importance as components of the resistanceresponse has been postulated (Rushton and Soms-sich, 1999). No effort was made to look specificallyfor systemic responses (van Wees et al., 2000), such as
induced systemic resistance/wound responses, a jas-monic acid-dependent response, or systemic ac-quired resistance, a salicylic acid-dependent re-sponse. Although some of the cDNA clones isolatedhave been associated with many plant stress re-sponses, including plant defense (specifically WRKY-18; Yu et al., 2001), as components of a signal trans-duction pathway, they may function as componentsof other plant processes. Fellbrich et al. (2002) dem-onstrated that induction of PR1 in Arabidopsis byNPP1, a homolog of NEP1 produced by Phytophthoraspecies, is salicylic acid-dependent. Salicylic acidplays a role in the regulation of plant processes otherthan plant defense, including leaf senescence (Morriset al., 2000). Similarly, Nep1 (Jennings et al., 2001)and NPP1 (Fellbrich et al., 2002) induce active oxygenproduction, and active oxygen can contribute to sus-ceptibility to disease (Tiedemannm, 1997; Baker andOrlandi, 1999) or resistance to disease (Baker andOrlandi, 1999). In many cases, it is the timing and/orlevel of gene expression that is characteristic of aresponse leading to compatibility or incompatibility(Graham and Graham, 1991; De Leon et al., 2002).
Using differential display, we were able to identifycDNA clones from multiple plant species potentiallyassociated with signal transduction pathways thatare responsive to Nep1, including calcium/calmod-ulin, DNA-binding/gene activation, lipid metabo-lism, reversible phosphorylation, and phytoalexinsbiosynthesis. Based on sequence homologies, thecDNA clones identified are in most cases closelyrelated to genes involved in plant defense and/orstress responses, including wounding, salicylic acid,drought, disease resistance, and senescence. Nep1induced ethylene biosynthesis in many differentplant species and Nep1 has been shown to induce1-aminocyclopropane-1-carboxylic acid synthase and1-aminocyclopropane-1-carboxylic acid oxidase tran-script accumulation in tobacco (Jennings et al., 2001).It is apparent from the transmission electron micro-graphs that chloroplasts and cuticles of all three spe-cies are rapidly altered in response to Nep1 treat-ment. Nep1 does not cause the same amount ofnecrosis in all the plant species it affects (Bailey,1995). Arabidopsis was much less sensitive to theNep1 rates and application methods used in thisstudy than were spotted knapweed and dandelion,although the types of damage caused by Nep1 weresimilar across the three plant species studied. Theprimary processes involved in the necrotic responsesof dicotyledonous plants to Nep1 appear to be simi-lar across species and to involve many differentgenes associated with plant responses to stress.
MATERIALS AND METHODS
Nep1 Purification
Nep1 was purified from culture filtrates of Fusarium oxysporum f. sp.erythroxyli grown for 6 d in Czapek-Dox broth plus 1% (w/v) casamino acids
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(Bailey, 1995). Protein concentration was determined by Bradford assay, andpurity was verified by SDS-PAGE (Bailey, 1995). Purified protein was storedin buffer (20 mm MES and 300 mm KCl, pH 5.0) at �20°C.
Plant Production
Seeds of spotted knapweed (Centaurea maculosa) were collected from localpopulations in Polo, IL, and seeds of dandelion (Taraxacum officionale) werecollected from local populations in Beltsville, MD. Seeds of Arabidopsis(wild type) and the two weed species were planted in 10.2-cm pots filledwith Scott’s Redi-earth, and plants were grown in ambient light and tem-perature conditions in a greenhouse. Plants were used in experiments 28 to32 d after planting.
Spray Application and Sampling
Nep1 was combined with 1,1,1,3,5,5,5-heptamethyltrisiloxanyl propyl-methoxy-poly[ethylene oxide] (Silwet-L77; Witco Corporation, Friendly,WV) for foliar spray applications to plants. Plants were treated with Nep1 (1or 5 �g mL�1) in 0.1% (v/v) Silwet-L77. Plants treated with Silwet-L77(0.1%, v/v) were included as a control. Foliar sprays were applied with asprayer (model 15; Binks, Glendale Heights, IL) at 15 psi and at a rate of 86mL m�2 (860.9 L ha�1). All sprays were applied between 10 am and 2 pmwhen stomata were fully open. Plants were maintained in greenhouseconditions until sampled. Leaves from Silwet-L77- (0.1%, v/v) and Nep1-treated (plus 0.1% [v/v] Silwet-L77) plants were collected 15 min, 1 h, and4 h after spray application, frozen in liquid nitrogen, and stored at �80°C.
Transmission Electron Microscopy
Leaf tissues for each plant species were collected 1 and 4 h after treatmentand were prepared for transmission electron microscopy. One leaf wassampled from three plants of each species for each treatment at each timepoint. Tissue squares (1 mm � 1 mm) treated with Silwet-L77 (0.1%, v/v) or1 �g mL�1 Nep1 (plus 0.1% [v/v] Silwet-L77) were dissected under 2.5%(w/v) glutaraldehyde/2% (w/v) paraformaldehyde in 50 mm PIPES buffer(pH 7.6) and were fixed for 24 h at 4°C (Karnovsky, 1965). Tissues werepostfixed in 2% (w/v) osmium tetroxide in 50 mm phosphate buffer. Sam-ples were dehydrated in an acetone series (30%–100% by 10% steps; 10 mineach step) and were infiltrated with Spurr’s (Spurr, 1969) epoxy resin (1:3,1:2, and 1:1 resin:acetone, each step overnight; three final changes of pureresin). Infiltrated tissues were placed in molds, and the resin was cured at80°C overnight.
Thin sections of embedded material were cut using a ultramicrotome(MT-5000; Sorvall, Kendro Laboratory Products, Newtown, CT) and werepicked up onto 200 mesh, formvar-coated nickel grids. Sections were stainedwith 2% (w/v) uranyl acetate for 10 min, rinsed, then stained with calcinedlead stain for 10 min. Sections were viewed and negatives were taken usinga transmission electron microscope (EM-10CA; Zeiss, Jena, Germany). Neg-atives were then scanned and saved as tiff files using a scanner (UMAXPowerlook 1100, UMAX Technologies, Inc., Dallas) connected to a computer(PowerMac G4; Apple Computers, Cupertino, CA).
Extraction of Total RNA
Total RNA was extracted from leaf tissues using a modified phenolextraction procedure (Goldsbrough et al., 1986; Kirby and Cook, 1967). RNAwas stored at a concentration of at least 2 �g �L�1 at �80°C.
Differential Display PCR
DNA was removed from total RNA by DNaseI treatment (MessageCleankit; Genhunter, Nashville, TN). Total RNA was isolated from each of thethree plant species 15, 60, or 240 min after treatment with 1 �g mL�1 Nep1(plus 0.1% [v/v] Silwet-L77). Total RNA isolated from plant tissues 240 minafter treatment with Silwet-L77 (0.1%, v/v) was included as the control.RNA was reverse transcribed, and the resulting cDNA was amplified byPCR using arbitrary 13 mers and anchored primers labeled with 5�-rhodamine (RNA Spectra Red kit 1; GenHunter). Eight arbitrary primers
(H-AP1 through H-AP8) were paired with three anchored primers for a totalof 24 primer combinations. Fluorescently labeled PCR product was sepa-rated on a 30 � 40 cm, 6% (w/v) denaturing polyacrylamide gel in Trisborate-EDTA buffer (60 W constant power for 3.5 h), and was imaged at 200�m resolution using a Cy3 setting (532 nm excitation laser, 555 nm emissionfilter with a 20-nm band pass filter) on a Typhoon 8600 Variable ModeImager (Molecular Dynamics/Amersham Biosciences, Piscataway, NJ). Dif-ferential display was carried out on replicate total RNA samples for eachplant species primer combination. Apparent differences in band intensitiesin control versus Nep1-treated tissues on the imaged gel were used to locatebands of interest. The opened gel was placed on top of a full-scale printoutof the fluorescent image of the gel, and the region containing the band ofinterest was excised and frozen at �20°C. The number of bands isolated was186 for spotted knapweed, 266 for dandelion, and 133 for Arabidopsis.Because replicate gels were run, ideally each band should have been isolatedtwice. Twenty-nine bands of interest were identified by careful comparisonsof display patterns and intensities between replicate gels (approximately10% if all the bands were duplicated) for use in this study. Of the 29 bands,15 were isolated from spotted knapweed, eight from dandelion, and sixfrom Arabidopsis.
Reamplification and Subcloning of cDNA Probes
Only DNA bands showing consistent differential expression on tworeplicate differential display gels were further processed. cDNA was elutedfrom excised gel slices and was reamplified by PCR using the primer setpreviously used for differential display PCR, but without fluorescentlylabeled anchored primer (RNA SpectraKit; GenHunter). cDNA probes weresubcloned using the PCR-Trap Cloning System (GenHunter). A subset of theprobes were reamplified, filter purified (Geneclean Turbo for PCR; Bio 101,Carlsbad, CA), and sequenced before subcloning as a means of tentativelyidentifying genes of potential interest based on sequence homology. Allsubcloned cDNA probes were sequenced to verify identifications madebefore subcloning (BigDye v. 3.0 dye terminator kit on an ABI 3100 Prism;Applied Biosystems, Piscataway, NJ).
Confirmation of Differential Gene ExpressionPatterns by Northern Blot
Twenty micrograms of total RNA was denatured in glyoxal/dimethylsulfoxide load dye at 50°C for 40 min (NorthernMax-Gly load dye; Ambion,Austin, TX) and was electrophoresed at 50 V for 4 h on a 1.2% (w/v) agarosegel containing 300 mm Bis-Tris, 100 mm PIPES, and 10 mm EDTA, at pH 8.0(1� BisTns-PIPES-EDTA buffer). RNA was attached to GeneScreen Plusmembrane (Perkin-Elmer Biosystems, Boston) by upward transfer in 10�SSC buffer. After crosslinking RNA to the membrane using the au-tocrosslink setting (UV Stratalinker 8600; Stratagene, La Jolla, CA), mem-branes were air-dried and stored between sheets of Whatman 3MM (What-man International, Ltd., Maidstone, Kent, UK) paper at �20°C. cDNAprobes were gel purified on a 1.5% (w/v) agarose gel (Geneclean II; Bio 101).For each probe, 35 ng of cDNA was labeled with [�-32P] dCTP using aRandom Primed DNA Labeling kit (Applied Biosystems, Foster City, CA)and gel filtered (Edge Gel Filtration Cartridges; Edge BioSystems, Gaithers-burg, MD). Blots were prehybridized in ExpressHyb solution (BD Bio-sciences/CLONTECH, Palo Alto, CA) at 68°C for 1 h.
Regardless of plant species, probes were initially hybridized to threereplicate blots, each containing total RNA from Nep1- (Nep1 1 �g mL�1,0.1% [v/v] Silwet-L77) and Silwet-L77-treated (0.1%, v/v) tissues for thethree species, spotted knapweed, dandelion, and Arabidopsis. Only probe/species combinations that showed significant hybridization in the studiesusing the 1 �g mL�1 Nep1 (plus 0.1% [v/v] Silwet-L77) rate were includedin studies using the 5 �g mL�1 Nep1 (plus 0.1% [v/v] Silwet-L77) rate.Radioactively labeled cDNA probes were denatured at 95°C to 100°C for 5min. The probe was added to 5 mL of fresh ExpressHyb solution, and blotswere incubated with continuous shaking at 68°C for 2 h. Blots were washedaccording to the ExpressHyb protocol, covered in plastic wrap, exposed tox-ray film at �70°C with two intensifying screens, and/or exposed to astorage phosphor screen (Molecular Dynamics/Amersham Biosciences),and imaged at 200 �m resolution on a Typhoon 8600 Variable Mode Imager(Molecular Dynamics/Amersham Biosciences). After imaging, the replicate
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1620 Plant Physiol. Vol. 132, 2003
blots were washed, scanned to verify that no probe remained, and probed asecond time with radioactively labeled 28S ribosomal cDNA probe.
Calculation of Band Volume Ratios
Blots probed with 28S ribosomal cDNA were imaged and band volumeswere calculated using ImageQuant Version 5.2c software (Molecular Dy-namics/Amersham Biosciences). To correct for variations in loading volumeon each replicate blot, the ratio of 28S ribosomal cDNA band volume foreach treatment combination (plant species/time/Nep1 rate) was deter-mined relative to the 15 min Silwet-L77 (thus arbitrarily set to one) controlsample. The band volumes for blots probed with cDNAs of interest werecorrected by multiplication by the appropriate 28S ribosomal cDNA ratio.For ease of presentation, the corrected band volumes were converted toratios relative to the 15-min Silwet-L77 (thus arbitrarily set to one) controlsample for each treatment combination on each replicate blot. The bandvolume ratios for each treatment combination were averaged using datafrom three replicate blots. The figures include the average band volumeratios (plus or minus 1 se) for each treatment combination in addition torepresentative autoradiograms. The three replicates for each treatment com-bination consisted of two samples from an initial experiment and a thirdsample from a subsequent experiment carried out approximately 6 monthsafter the initial experiment.
Received December 27, 2002; returned for revision January 30, 2003; ac-cepted February 16, 2003.
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